Roundabout homolog 4 (ROBO4), a protein of 110 kDa in molecular weight, has a single-pass transmembrane structure (Non Patent Literature 1) and is known to suppress angiogenesis through the binding to a known angiogenesis suppressor Slit homolog 2 (Slit2) (Patent Literature 1 and Non Patent Literature 2). Slit2 has been reported to suppress the migration of HUVEC promoted by a vascular endothelial growth factor (VEGF) and has also been reported to suppress the promotion of cell migration by VEGF or bFGF for ROBO4 gene-transfected vascular endothelial cells (hereinafter, the vascular endothelial cell is also referred to as “EC”) compared with empty vector-transfected EC (Patent Literature 1 and Non Patent Literatures 2 to 4).
Moreover, it has been reported that the suppressive effect of Slit2 on the promotion of cell migration, promotion of lumen formation, or increase in permeability by VEGF is observed in EC derived from a wild-type mouse or EC transfected with control siRNA, but not observed in EC derived from a ROBO4 gene-knockout mouse or EC transfected with siRNA to knock-down the ROBO4 gene (Patent Literature 2 and Non Patent Literatures 4 to 6). Furthermore, it has been reported that Slit2 suppresses, via ROBO4, angiogenesis or increase in vascular permeability in mouse models with laser-induced choroidal neovascularization or oxygen-induced retinopathy, which are animal disease models with exudative age-related macular degeneration or diabetic retinopathy (Patent Literature 2 and Non Patent Literature 4).
In spite of these findings, there are also reports showing that ROBO4 does not bind to Slit2 (Non Patent Literature 9 and Patent Literature 5). It has also been reported as to its functions that ROBO4 participates in the promotion of angiogenesis rather than the suppression of angiogenesis, because the migration or lumen formation of ROBO4 gene-knockout EC is inhibited (Non Patent Literatures 10 and 11).
In clinical practice, ROBO4 has been reported to be highly expressed in intratumoral vessels in liver metastasis from colon cancer, ganglioglioma, bladder cancer, breast cancer, metastatic melanoma, kidney cancer, lung cancer, liver cancer, or colon cancer (Patent Literature 3 and Non Patent Literatures 1, 3, and 7). Moreover, ROBO4 has been reported to be also expressed in blood vessels in the fibrovascular membranes of proliferative diabetic retinopathy patients (Non Patent Literature 8). As such, ROBO4 is expressed in vascular endothelial cells, particularly, endothelial cells in blood vessels newly formed in a pathological condition. This may suggest a pathological angiogenesis resulting from the high expression of ROBO4, but may also suggest the compensatory expression of ROBO4 for suppressing pathological angiogenesis.
As described above, ROBO4 is involved in an anti-angiogenesis effect. Thus, an antibody against ROBO4 and a functional fragment thereof are presumably useful in the treatment of a disease involving angiogenesis. However, it is uncertain whether either an agonistic or antagonistic antibody against ROBO4 suppresses or promotes angiogenesis.
Antibodies described in EP Patent No. 1,565,491 (Patent Literature 4) and WO2008/100805 (Patent Literature 5) are known as the antibody against ROBO4 (hereinafter, referred to as an “anti-ROBO4 antibody”). But none of these antibodies does not show a suppressive or inhibitory effect on angiogenesis in vivo.